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J. Biol. Chem., Vol. 281, Issue 46, 35413-35424, November 17, 2006
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From the
Department of Microbiology and Immunology, and Sheldon Biotechnology Centre, McGill University, Montreal, Quebec H3A 2B4, Canada
Received for publication, August 9, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The ferrichrome transport system consists of four proteins (FhuA, FhuB, FhuC, and FhuD) expressed by Gram-negative bacteria. The FhuA protein consists of two domains as follows: an N-terminal globular cork domain is enclosed by a 22-stranded C-terminal 
-barrel domain (2, 3). Connections between -strands in the barrel domain are such that long loops participating in ferrichrome binding are exposed to the extracellular environment; short turns are exposed to the periplasm. Uptake of ferrichrome is a TonB-dependent process, mediated by contacts between TonB and the OM receptor FhuA. TonB is elongated and has three domains as follows: an N-terminal domain anchored in the CM, an intermediate domain containing Pro-Glu and Pro-Lys repeats, and a globular C-terminal domain with a central -sheet and two 
-helices. To date, structural data are only available for the C-terminal domain (4-6). We recently solved the crystal structure of the 1:1 TonB-FhuA complex (7). The C-terminal domain of TonB makes extensive contacts with the N-terminal consensus Ton box of FhuA, as well as residues Ala-26 and Glu-56 of the cork domain and with periplasmic turns 8 and 10. These contacts orient TonB such that it may mediate conformational disruption of the internal cork domain of FhuA, allowing for passage of siderophore into the periplasm.
Although recent structural and biophysical data have clarified initial steps of the siderophore transport cycle involving TonB-receptor interactions, little is known about the fate of the siderophore once transported into the periplasm. Specifically, the molecular mechanisms of siderophore transport from periplasm to cytoplasm are largely uncharacterized. FhuD in the periplasm binds the hydroxamate siderophores ferrichrome, coprogen, and aerobactin (8). Loss of FhuD function in vivo prevented growth of Escherichia coli under iron-limiting conditions when ferrichrome, coprogen, or aerobactin were used as the sole iron source, suggesting that FhuD is a necessary component of the hydroxamate siderophore transport system (8). FhuD was reported to interact with regions of the CM-embedded permease FhuB. Interactions between FhuD and FhuB have been demonstrated by cross-linking studies, protease protection assays (9), and enzyme-linked immunosorbent assay (10). Interaction of FhuB with FhuD is apparently independent of siderophore binding by FhuD (9). Taken together, these results suggest that FhuD functions as a carrier protein; ferrichrome released from the OM receptor is delivered by FhuD to the permease. The integral membrane protein FhuB then translocates the siderophore into the cytoplasm mediated by ATP hydrolysis of FhuC (11).
The crystal structure of FhuD in complex with gallichrome, a ferrichrome analogue, has been reported (12), as well as structures of FhuD in complex with albomycin, coprogen, and Desferal® (13). The fold of this 32-kDa protein is bilobal; globular N- and C-terminal domains are connected by a long -helix that confers rigidity to the protein. The siderophore-binding site residing in a shallow cleft between the two lobes is hydrophobic, having predominantly aromatic residues. Siderophore binds to FhuD through both hydrophobic and hydrophilic interactions. Methylene carbon atoms in the siderophore form hydrophobic interactions with numerous aromatic FhuD residues in the binding cleft. Hydrogen bonds are formed between hydroxamate groups of the siderophore and FhuD residues Arg-84 and Tyr-106. A hydrogen bond with the siderophore is also formed with FhuD residues Asn-215 and Ser-219 through an intermediate water molecule. The overall fold of FhuD is similar to that of BtuF (14, 15), the periplasmic cobalamin-binding protein of E. coli. Periplasmic metal-binding proteins TroA (16) and PsaA (17) are also structurally related to FhuD. These proteins share a fold distinct from those of classical periplasmic proteins such as maltose-binding protein (18). However, unlike maltose-binding protein, FhuD does not exhibit gross conformational rearrangements upon ligand binding. The linker connecting the N-terminal and C-terminal domains in FhuD is a kinked -helix that crosses these domains only once. The structure of FhuD and the hydrophobicity of the siderophore-binding site suggest that large scale opening and closing of the binding site does not occur upon siderophore binding and release (18). Molecular dynamics simulations also suggest that FhuD is conformationally rigid but that subtle conformational differences in the C-terminal domain between the apo- and holo-forms may be sufficient for discrimination by FhuB (19).
What molecular events result in capture of ferrichrome by FhuD following its TonB-dependent release from FhuA? Given the apparent weak affinity (1 µM) of ferrichrome for FhuD (9), binding is unlikely to be a diffusion-governed process. Efficiency of siderophore capture would be enhanced by positioning a binding protein proximal to the lumen of the OM receptor. This organization would promote direct transfer of ferrichrome from FhuA to FhuD. Here we report the first biophysical evidence that TonB specifically interacts with FhuD. Discrete regions of protein-protein interactions on the surfaces of both FhuD and TonB were identified by phage display. Interactions were confirmed by dynamic light scattering, fluorescence spectroscopy, and surface plasmon resonance. Our results suggest that siderophore released from FhuA during the transport cycle is transferred to FhuD via a coordinated transfer mechanism mediated by TonB. Hence, TonB would act as a periplasm-spanning scaffold, directly connecting siderophore transport events between the OM and CM.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Chemicals and Reagents—5-Bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal) and isopropyl -D-thiogalactopyranoside were purchased from BioVectra (Charlottetown, Prince Edward Island, Canada). Protein-grade Tween 20 was purchased from Calbiochem. Antibiotics were purchased from Sigma. Nickel-nitrilotriacetic acid resin used for protein purifications was purchased from Qiagen. The reducing agent tris-(2-carboxyethyl)phosphine and fluorescent dyes 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthaline-1-sulfonic acid (AEDANS) and 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) were purchased from Invitrogen.
Protein Purification—TonB and Cys-TonB were purified as described previously (20). To purify overexpressed FhuD or FhuD T181C, cell pellets were suspended in 50 ml of buffer A containing 50 mM Tris, pH 8.2, 150 mM NaCl, and 5 mM imidazole plus one Complete mini EDTA-free protease inhibitor mixture tablet (Roche Applied Science); 0.16 mg/ml lysozyme and 16 µM phenylmethylsulfonyl fluoride were then added. Cells were shaken at room temperature for 30 min, followed by addition of 0.04 mg/ml DNase, 0.04 mg/ml RNase, and an additional 16 µM phenylmethylsulfonyl fluoride. To lyse bacteria, cells were passed twice through an Emulsiflex-C5 (Avestin, Ottawa, Ontario, Canada). Cell lysate was centrifuged (27,000 x g, 4 °C) for 50 min and filtered through 0.45-µm syringe filters. Filtered cell extracts containing FhuD or FhuD T181C were applied to nickel-nitrilotriacetic acid resin equilibrated with buffer A. FhuDs were eluted with 50 mM Tris, pH 8.2, containing 125 mM imidazole, pooled, and applied to a POROS HQ 20 anion exchange column (Applied Biosystems). Bound proteins were washed with 50 mM Tris, pH 8.2, containing 125 mM imidazole, eluted with 160 mM NaCl, and applied inline to a POROS MC 20 column (Applied Biosystems). After extensive washing, proteins were eluted with 50 mM Tris, pH 8.2, and 120 mM imidazole and applied to a second POROS HQ 20 column, washed as described above, and eluted with 180 mM NaCl in 50 mM Tris, pH 8.2. Purified proteins were dialyzed in a 24,000 Mr cut-off dialysis membrane (SpectraPor) for 16 h at 4 °C in 100 mM Hepes, 150 mM NaCl, pH 7.4. Homogeneity of FhuDs was confirmed by SDS-PAGE and silver staining of 750 ng of total protein. Concentrations of protein were determined by either a Bradford or BCA assay using bovine serum albumin as standard.
Phage Display—Phage panning against TonB as target was described previously (24). Purified FhuD was diluted to 100 µg/ml in TBS (Tris-buffered saline: 50 mM Tris, pH 7.5, 150 mM NaCl), and 150 µl of protein was adsorbed to a polystyrene microtiter plate (Nunc Maxisorp). Plates coated with immobilized FhuD were incubated for 16 h at 4 °C followed by blocking (2 h at 37 °C) with TBS containing 5 mg/ml bovine serum albumin. The unselected phage library (New England Biolabs) was then added. Phage panning, clone isolation, DNA sequencing, and bioinformatic analyses were performed as described previously (24).
Dynamic Light Scattering—Light scattering was measured from purified TonB and FhuD dialyzed twice (18 h, 4 °C) in 100 mM Hepes, pH 7.4, containing 150 mM NaCl. Purified [Fhu switch-MBP] fusion protein (24) (containing FhuA residues 21AWGPAAT27 fused to the N terminus of maltose-binding protein) and BSA were dialyzed against the same buffer and used in DLS measurements as positive and negative controls, respectively. TonB (4.0 µM) and FhuD (3.0 µM) were separately analyzed as discrete scattering species. Similarly, BSA (1.5 µM) and [Fhu switch-MBP] (2.5 µM) were analyzed separately. For a 1:1 molar ratio of TonB to FhuD, each protein at 1.7 µM was mixed prior to recording DLS readings. For 1:1 mixtures of TonB with BSA or with [Fhu switch-MBP], proteins were each at 1.0 µM. Protein mixtures were incubated for 30 min at room temperature prior to centrifugation and analysis. Data acquisition was performed in a 12-µl quartz cuvette at 20 °C using a temperature-controlled DynaPro E-50-830 dynamic light scattering instrument (Protein Solutions, Charlottesville, VA). The scattering signal was measured at a wavelength of 824.9 nm and an angle of 90°. Data were collected for 7000 s with a 10-s averaging time and replicated with two independent protein preparations. From the Dynamics version 6.3.18 software (Protein Solutions, Charlottesville, VA), data were filtered (base line <1.01 and sum of squares <500) before exporting to Sedfit version 9.3. Analyses of hydrodynamic radii (Rh) were performed using the continuous intensity distribution model (25) in Sedfit at a resolution of 100 for radii between 1 and 50 nm. Buffer densities and viscosities were set to 1.00442 and 0.01065, respectively, as determined by Sednterp version 1.08. All values of Rh from the Dynamics software exhibited less than 14% polydispersity, except for the TonB-FhuD mixture and [Fhu switch-MBP] (21 and 19%, respectively).
For Sedfit analyses of discrete noninteracting species (DNS), autocorrelation data sets were imported from the Dynamics software package and fit to a single species field autocorrelation function. Values of s for TonB were determined previously (20). Using analytical ultracentrifugation, we determined by sedimentation velocity experiments sedimentation coefficients for FhuD and the TonB-FhuD complex, 2.27 s and 3.5 s, respectively. From literature reports, s values for MBP (26) and BSA (27) were obtained. All were constrained in DNS analyses. Molecular mass values for discrete scattering species, either uncomplexed TonB, uncomplexed FhuD, or 1:1 heterocomplexes, were initially set to predicted values and then refined by nonlinear regression until r.m.s.d. errors were minimized. In addition to proteins TonB, FhuD, [Fhu switch-MBP], or complexes formed by these proteins, two scattering species were observed; the Dynamics program predicted these uncharacterized species to have hydrodynamic radii of 1 and 100 nm respectively. Hydrodynamic parameters for these species were factored into DLS analyses to optimize fits to the autocorrelation function.
Fluorescence Spectroscopy—The fluorescent dye AEDANS was conjugated to FhuD and FhuD T181C in a reaction buffer of 100 mM Hepes, pH 7.4, 150 mM NaCl. Following reduction of disulfide bonds with a 10-fold molar excess of tris-(2-carboxyethyl)phosphine, dye was added to a 10-fold molar excess. Conjugation proceeded in the dark with stirring for 4 h at room temperature. Reactions were quenched by addition of -mercaptoethanol. Excess label was removed by exhaustive dialysis against four 1-liter changes of 100 mM Hepes, pH 7.4, containing 150 mM NaCl in the dark at 4 °C. After dialysis, free dye was present at picomolar concentrations. Conjugates were then centrifuged at 18,000 x g for 30 min at 4 °C. Labeled proteins were stored at 4 °C in the dark. Conjugation of FhuD T181C with the dye MDCC was performed as described above except that MDCC was dissolved in Me2SO prior to its addition to protein. Efficiency of labeling (mol dye:mol protein) was calculated from absorption data using the following tabulated (Invitrogen) molar extinction coefficients: 5700 M-1 cm-1 at 336 nm for AEDANS and 50,000 M-1 cm-1 at 419 nm for MDCC and from protein concentrations as determined by protein assays.
Fluorescence data were collected with a Varian Cary Eclipse fluorescence spectrophotometer. Emission spectra were recorded at excitation and emission wavelengths of 280 and 340 nm, respectively, for intrinsic fluorescence measurements; at 336 and 490 nm, respectively, for AEDANS-labeled FhuD and FhuD T181C; and at 419 and 466 nm, respectively, for MDCC-labeled FhuD T181C. Excitation and emission slits were set between 2.5 and 5 nm and 5 and 10 nm, respectively. Measurements were taken in triplicate at 20 °C. Data were corrected for changes in fluorescence intensity attributed to dilution of protein and the minimal fluorescence contributions of Fcn, TonB, and buffer (100 mM Hepes, pH 7.4, 150 mM NaCl).
Binding of Fcn to either FhuD (1.5 µM) or FhuD T181C (0.5 µM) was monitored by recording the fluorescence emission after additions of Fcn up to a 10-fold molar excess. For each data point, Fcn was added from a stock solution, and after 3 min of incubation, the change in fluorescence was recorded. Titrations of labeled conjugates with either Fcn or TonB were conducted in an identical manner. Fluorescence quenching was expressed as the percentage decrease in fluorescence upon ligand addition compared with the theoretical maximum whereby quenching would result in complete loss of fluorescence. Data were fit (Sigmaplot) to an equation describing a rectangular hyperbola using the single binding site model or to a sum of two hyperbolics using the model that describes two independent binding sites.
Surface Plasmon Resonance (SPR)—Binding interactions between TonB and FhuD or between Cys-TonB and FhuD were examined in real time using BIAcore 2000/3000 instrumentation with research grade CM4 sensor chips (BIAcore AB, Uppsala, Sweden). Experiments were performed in triplicate at 25 °C using filtered (0.2 µm) and degassed HBS-ET (50 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Tween 20). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide, and 2-(2-pyridinyldithio)ethaneamine were from BIAcore AB. Protein grade detergents (10% Tween 20, 10% Triton X-100, 30% Empigen) were from Calbiochem. All other chemicals were reagent grade quality.
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120 RU were bound. Finally, three injections (20 µl) of freshly prepared solution III (50 mM L-cysteine in 0.1 M sodium formate, pH 4.3, containing 1 M NaCl) deactivated excess reactive groups and removed any nonspecifically bound ligand. Coupling efficiencies were typically 50%. Reference surfaces were prepared in a similar manner without any ligand addition.
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2 values below 1. Multicomponent SPR analyses between FhuA, TonB, and FhuD were performed. Initially, amine-coupled TonB surfaces (250 RU) or thiol-coupled Cys-TonB surfaces (100 RU) were prepared. Then, either a TonB-FhuA or a TonB-FhuD binary complex was formed by injecting each analyte at 50 µl/min. By injecting FhuD over TonB-FhuA complexes or by injecting FhuA over TonB-FhuD complexes, ternary complex formation was assessed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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17 x 17 Å that is formed at the base of the siderophore binding pocket. In addition to loop 23, region IV also includes residues from helix 13 near the C terminus of FhuD. Although the surface-exposed residues in region III could form a potential TonB-binding landscape with regions I, II, and IV, residues 81-88 in loop 3 preclude formation of such a continuum. No FhuD residues within this interval were identified by our phage display analysis, suggesting that loop 3 is not TonB-binding. If there was a continuous landscape of all four regions, it would require a displacement of loop 3 from FhuD to accommodate TonB binding. Recent molecular dynamics simulations on the FhuD structure (19) indicated that the C-terminal domain of FhuD has more overall mobility than the N-terminal domain. However, within the relatively static N-terminal domain, loop 3 was observed to be the most mobile region.
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Both our phage display outcomes (24) and the x-ray crystallographic structure (7) of the TonB-FhuA complex demonstrated that residues in the switch helix region of FhuA interact directly with TonB. DLS analyses of [Fhu switch-MBP] fusion protein uncomplexed or in complex with TonB yielded results (Table 5, rows 4 and 5) in agreement with these previous analyses. The refined molecular mass for uncomplexed [Fhu switch-MBP] agreed with its predicted molecular mass. When [Fhu switch-MBP] was mixed with TonB in a 1:1 molar ratio, an abundant scattering species of 61 kDa was observed, consistent with formation of a 1:1 heterocomplex between TonB and the fusion protein.
Detection of a TonB-FhuD Complex by Fluorescence Spectroscopy—Guided by the TonB-binding surface on FhuD that was predicted from phage display, we generated a mutant, FhuD T181C, to which were conjugated thiol-reactive probes capable of reporting changes in local environment. This mutant was assessed for its binding of ligand and for its binding of TonB. Rohrbach et al. (9) observed that addition of ferrichrome caused marked quenching of the intrinsic fluorescence of FhuD and used this observation to quantify binding of various ligands. We extended this feature to measure the ligand binding capacity of FhuD T181C. Addition of Fcn to either FhuD or to FhuD T181C caused substantial decreases in emission maxima. Binding curves were generated by plotting the percentage decrease in fluorescence as a function of Fcn added (Fig. 5A). Fits of these data (Table 6) to a single binding site model yielded similar apparent dissociation constants (KD(app)) of either 1.2 ± 0.2 µM (FhuD) or 0.6 ± 0.2 µM (FhuD T181C), in agreement with the previously reported (9) KD of 1 µM. Our results establish that the T181C mutation does not compromise ligand binding.
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1 mol of conjugate for every 3 mol of protein. This outcome likely resulted from labeling conditions. MDCC must be dissolved in Me2SO; addition of this labeling solution to FhuD resulted in slight precipitation compared with the aqueous labeling conditions used with AEDANS, in which no precipitation was observed.
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FhuD T181C labeled with either AEDANS or MDCC demonstrated marked changes in fluorescence when mixed with TonB. Upon addition of TonB to labeled conjugates, quenching was observed (Fig. 6), demonstrating that the presence of TonB altered the environment of the probe. These changes were not observed when labeled FhuD T181C was mixed with an excess of BSA (data not shown). At low micromolar amounts of added TonB, saturation was achieved. Fits of these data to a single binding site model generated values as listed in Table 6. The KD(app) value for the TonB-FhuD complex was 0.31 ± 0.05 µM for AEDANS-labeled FhuD T181C and 0.27 ± 0.04 µM for MDCC-labeled FhuD T181C. Prior saturation of the FhuD-binding site with Fcn did not affect binding of TonB (Fig. 6) because similar dissociation constants (Table 6) were determined despite occupancy.
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Alteration of labeling conditions affected conjugation efficiencies. By labeling FhuD T181C overnight with AEDANS at 4 °C, 2 mol of label incorporated for every mol of protein. FhuD contains a single endogenous Cys-237. Based upon the x-ray crystal structure, this residue is not expected to be solventexposed. To determine whether Cys-237 was reactive to conjugation, we labeled FhuD by using the conditions above; unexpectedly, FhuD was labeled. Cys-237 localizes outside the phage display-identified TonB-binding surfaces. Given the reactivity of Cys-237 toward conjugation, we used this conjugate to exclude the possibility that TonB binds to regions other than those implicated by phage display. Addition of TonB to FhuD-AEDANS caused an insignificant increase in the fluorescence emission of AEDANS (data not shown). Given this outcome that contrasts with FhuD T181C-AEDANS, we consider that Cys-237 is not part of a TonB-binding environment. Our conclusion is that TonB binding localizes to regions on FhuD that were identified by phage display.
Detection of a TonB-FhuD Complex by Surface Plasmon Resonance—Our previous use of SPR technology quantified binding of FhuA to immobilized TonB (20). Given the outcomes of this experimental design, we adapted its use for the study of TonB-FhuD interactions. Dose-dependent binding of FhuD to amine- and thiol-coupled TonB surfaces was observed (Fig. 7). Purified FhuA (0.1-1 µM) also bound to immobilized TonB as a positive control; FhuD binding (0.5 µM) was unaltered in the presence of BSA (0.5 µM) as a competitor (data not shown). To improve kinetic analyses, a homogeneous presentation of a lower density thiol-coupled Cys-TonB surface was utilized. A high affinity interaction (KD 20 nM) between FhuD and TonB was determined by SPR using a single binding site model. This interaction was characterized by slow association (ka, 2 x 104 M-1 s-1) and slow dissociation rates (kd, 4 x 10-4 s-1). Presence of the siderophore Fcn did not significantly alter the affinity between FhuD and TonB by SPR (Fig. 7B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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4,000 copies of the ferric enterobactin periplasmic binding protein FepB in E. coli, a low value compared with other periplasmic binding proteins such as those involved in sugar or amino acid transport. Köster and Braun (8) demonstrated that chromosomally encoded FhuD was undetectable from silver-stained SDS-polyacrylamide gels of periplasmic extracts. Taken together with the modest KD value of 1.0 µM (9), these data suggest that diffusion alone would be insufficient to account for unidirectional siderophore transport. In addition to siderophore capture, diffusion-governed docking of siderophore-bound FhuD to the CM permease may be an inefficient process.
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Interactions between TonB and FhuD are multidimensional. By DLS, we identified a 1:1 TonB-FhuD complex. Fluorescence spectroscopy indicated an apparent affinity for this complex to lie within the mid-nanomolar range. This affinity contrasts with the low nanomolar range determined by SPR. Our current SPR studies for FhuD-TonB are similar to the previous experimental design used to monitor the TonB-FhuA kinetics in real time (20). The affinity range predicted for TonB-FhuD interactions by SPR (low nanomolar) is consistent with the previously reported range (35) of TonB-FhuA interactions also by SPR. The differences in reported affinities between fluorescence and SPR may be attributed to solution phase versus immobilized systems or to buffer requirements of the different technologies. Given these differences, our data indicate that the affinity between TonB and FhuD lies somewhere in the low to mid-nanomolar range.
Significantly, the presence of Fcn did not alter the affinity between TonB and FhuD; there was no evidence for competition. However, the TonB-binding surface on FhuD as identified by phage display both overlaps the siderophore-binding site and extends beyond it; competition would not be expected. Furthermore, there is no evidence to suggest large conformational changes in FhuD upon binding siderophore (19). In the siderophore-bound state, FhuD probably maintains a rigid backbone; this rigidity would not influence binding of TonB. One implication of these findings is that interactions between siderophore and FhuD are distinct from interactions between TonB and FhuD. It remains to be determined whether siderophore can still bind to FhuD when complexed with TonB or whether bound TonB prevents siderophore binding through occlusion of siderophore-binding site for FhuD.
A regulated mechanism of siderophore transport would involve coordination of protein-protein interactions that would facilitate direct transfer of siderophore among protein partners. For such a mechanism, siderophore transfer would follow a sequence of directed exchanges from OM receptor, to periplasmic binding protein, to CM permease. Directed transfer of this sort would require a scaffold whereby protein-protein interactions drive spatial and temporal localization of the periplasmic binding protein to regions involved in siderophore translocation activities. The dynamic nature by which TonB cycles or changes its conformation during energy transduction offers itself as an ideal candidate to fulfill this role.
Our SPR data indicated formation of a ternary FhuA-TonB-FhuD complex. This finding, taken together with the recent structural determinations of TonB bound to FhuA (7) and to BtuB (36), underscores the possibility of such a complex. It would position a periplasmic binding protein to accept siderophore immediately after its translocation through a TonB-dependent OM receptor. Examination of the TonB-FhuA interaction surface provides a means to evaluate residues involved in the protein-protein interaction. In the TonB-FhuA crystal structure, residues N-terminal to Arg-158 on TonB were not resolved and may not participate in the TonB-FhuA interface. The FhuD-interacting residues on TonB that were predicted by phage display (Fig. 3C, region III) localize immediately N-terminal to TonB Arg-158. We infer that these residues are poised to bind FhuD in a FhuA-TonB-FhuD ternary complex.
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The precise sequence of these events has yet to be established. Our data cannot distinguish whether FhuD remains bound to TonB during the energy transduction cycle or if binding is a transient event. One possibility is that FhuD binds and dissociates as a function of the TonB energy transduction cycle, perhaps resulting from the -strand exchange that TonB is known to undergo (4, 5, 7, 30, 36). Prolonged association of FhuD with TonB seems unlikely as FhuD must ultimately deliver siderophore to FhuB/C. It is intriguing that the most extreme N-terminal FhuD-binding surface on TonB is at a region close to probable contact sites with ExbB/ExbD. Such placement may be a means by which TonB directs transfer of FhuD from the OM to CM as TonB disengages FhuA. This mechanism would ensure that FhuD samples a space proximal to the CM, thereby enhancing the probability of encountering FhuB. Previous reports have indicated (14, 37) that interactions between the FhuD homologue BtuF and its cognate transporter BtuC/D are virtually irreversible. This observation remains to be clarified in light of our experimental evidence, which for the first time identifies TonB as a binding partner for periplasmic binding proteins. We advocate a mechanism favoring transient associations of periplasmic siderophore-binding proteins with TonB and with their cognate CM transporters at discrete steps during the siderophore transport cycle.
We previously used phage display in concert with biophysical methods to map unambiguously the network of protein-protein interactions that occur at the TonB-FhuA interface. These outcomes were recently confirmed by the x-ray structure of the TonB-FhuA complex. This strategy is now extended to identify interfaces involved in TonB-FhuD interactions. Such interactions may serve to coordinate spatial and temporal localization of periplasmic binding proteins to environments involved in siderophore uptake. Given the diversity of components among different siderophore transport systems, we propose that in addition to its role as energy transducer, TonB acts as a unifying element, a scaffold to regulate the unidirectional flow of iron-bound siderophore from the OM to the CM.
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FOOTNOTES |
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1 Recipient of the McGill Graduate Studies Fellowship and an F. C. Harrison Fellowship.
2 Recipient of a Natural Sciences and Engineering Research Council (Canada) Undergraduate Student Research Award.
3 Recipient of a postgraduate scholarship.
4 Trainee from theÉcole Supérieure de Biotechnologie, Strasbourg, France. Present address: Dept. of Life Science, Imperial College, London, UK.
5 Recipient of a traveling award for research training from the National Health and Medical Research Council (Australia). Present address: Dept. of Microbiology and Immunology, The University of Melbourne, Parkville, Australia.
6 Present address: Dept. of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B 1R6, Canada.
7 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, McGill University, 3775 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: 514-398-3929; Fax: 514-398-7052; E-mail: james.coulton{at}mcgill.ca.
8 The abbreviations used are: OM, outer membrane; CM, cytoplasmic membrane; Fcn, ferricrocin; DLS, dynamic light scattering; SPR, surface plasmon resonance; r.m.s.d., root mean square deviation; BSA, bovine serum albumin; MBP, maltose-binding protein; AEDANS, 5-((((2-iodoacetyl)amino)-ethyl)amino)naphthaline-1-sulfonic acid; RU, response units; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin; PDB, Protein Data Bank; DNS, discrete noninteracting species; Rh, hydrodynamic radius.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) and FhuD T181C (
) were titrated with the indicated amounts of Fcn; quenching of intrinsic fluorescence is plotted as a function of Fcn concentration. B, binding of Fcn to AEDANS-labeled FhuD T181C (
). Proteins were titrated with Fcn, and quenching of probe fluorescence was plotted as a function of Fcn added. Error bars in A and B represent the standard deviation from three independent experiments. Lines through data indicate best fits to a single binding site model as determined with Sigmaplot.
